The purpose of this project is to isolate genes encoding the heavy chains of nonmuscle myosins and to use these genes as tools to investigate myosin structure/function relationships and the in vivo functions of nonmuscle myosins. Using molecular cloning techniques, we previously isolated from the soil amoeba Acanthamoeba castellanii the heavy chain genes for myosin II (a nonmuscle myosin possessing conventional structure) and myosin IB (an unusual, low-molecular weight, non-filamentous nonmuscle myosin). In this report we describe the cloning and characterization of two additional amoeba myosin heavy chain genes and our initial efforts at cloning a gene encoding a vertebrate form of myosin I. The significance of this work is that (i) the deduced protein sequences are of great value in furthering our understanding of the structural and functional properties of these myosins and (ii) by using the tools of molecular biology we can approach the study of these proteins in novel ways which are not possible using the classical techniques of protein chemistry. For example, we can dissect at the molecular level the mechanisms of enzymatic regulation using site-directed mutagenesis to alter the myosin molecules and we can examine the physiological roles of these myosins by reintroducing the genes back into cells. Both of these approaches are currently underway.